Identification of GABA receptors in chick cornea.

Purpose The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question.

The cornea, as the eye's first refracting surface, plays an important function in vision, is highly innervated and has precise mechanisms for maintaining optical clarity. The chick cornea has been suggested to be an excellent model for the study of wound healing, scar tissue formation and neuronal re-innervation [1] and thus it is important to learn more about chick corneal anatomy and physiology. Many neurotransmitter receptors have been shown to be present in the cornea, e.g., muscarinic [2], melatonin [3], and dopamine receptors [4], but others appear to have not been studied to any siginificant degree (e.g., γ-aminobutyric acid [GABA] receptors). In the literature there is a sole report of GABA receptor expression in cultured human stem cells [5].
Knowledge regarding the distribution of GABA receptors in the eye is very limited and information on GABA receptors in the cornea even more sparse. GABAA [6][7][8], GABAB [9][10][11], and GABAC [12] receptors have been reported in the retina. We have recently shown that rho1 GABAC receptors are present in chick sclera [15]. A small number of cultured human corneal stem cells have been shown to exhibit GABAA receptor immunoreactivity [5]. We found no reports of studies of GABA receptor expression in any animal species, although there is a report of GABA modification of the rabbit corneal endothelial fluid pump [16] and an observation of activity of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), in rat cornea [17].
A range of other neurotransmitter receptors have been reported in corneal tissues of a diverse animal species [2][3][4]18] and human tissue [19,20]. The muscarinic receptor subtypes M1 and M2 are expressed in bovine corneal epithelial cells [2]. In the rabbit cornea, D1 and D2 dopamine receptors have been localized to both epithelial and endothelial layers [4]. In the mouse cornea, retinoic acid (RA) receptors have been detected in the epithelium and stroma [18]. In the Xenopus laevis eye, melatonin receptors have been visualized in the corneal epithelium, fibroblasts, and endothelium [3]. In the human cornea, muscarinic M1-5 receptors have been observed on corneal epithelial cells [19], and M2, M4, and M5 are expressed in corneal endothelium [20].
The fact that human corneal stem cells express GABAA receptors [5], that there are reports of some GABA actions in the cornea of animal species [16,17], that GABA receptors are present in a range of non-neuronal tissues [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36], and that the chick sclera possess GABAC receptors [15], lead to the hypothesis that GABA receptors may be present in chick cornea. The aim of this study was to determine if GABAA, GABAB, and/or GABAC receptors are located in the chick cornea. The secondary aim was to determine if expression occurred in the corneal endothelium, stroma or epithelium.

METHODS
This research comprised: (1) study of the expression of alpha1 GABAA, GABAB, and rho1 GABAC receptors mRNA in chick cornea, and (2) identification and distribution of alpha1 GABAA, GABAB, and rho1 GABAC receptors protein in chick cornea.
Animals and tissue preparation: Animals and tissue were prepared as previously described [15]. Twenty-five 12-dayold White Leghorn cockerels (Gallus gallus) were obtained from Jinan Spafury Poultry Farms (Jinan, China). Animals were administered a lethal dose of pentobarbital sodium (3%, 5ml/kg; Beijing Chemistry Com., Beijing, China) and the eyes enucleated. Eyes (n=5, 1 eye from 5 chicks) for immunohistochemistry were placed whole into microtubes, snap frozen in liquid nitrogen and stored at −80 °C. Eyes for real time PCR (n=5 different samples, 4 eyes of 2 chicks were used as 1 sample, 20 eyes of 10 chicks were used) and western Blot (n=5 different samples, 4 eyes of 2 chicks were used as 1 sample, 20 eyes of 10 chicks were used) were placed on a cold plate (8 °C) and the corneal tissue was removed along the limbus, and was separated from the sclera, conjunctiva, and iris using surgical scissors and forceps under a dissection scope. Corneal tissues from 4 eyes of 2 chicks formed one sample and were placed in separate microtubes, snap frozen in liquid nitrogen and stored at −80 °C until processing. Retina from these eyes were obtained and used as the positive control; the retina has been previously shown to possess alpha1 GABAA [6][7][8], GABAB [9][10][11], and rho1 GABAC [12] receptors. Real-time PCR: Real-time PCR was performed as previously described [15,37] with minor modification according to the manufacturer's instructions. Total RNA was extracted from chick cornea and retina using Trizol Reagent (Invitrogen, Carlsbad, CA). RNA concentration and purity were determined at an optical density ratio of 260:280 using a spectrophotometer. CDNAs (cDNAs) were synthesized with 1 μg of total RNA, 1 μl random primer,1 μl dNTPs, 2 μl DTT, and 200 U MMLV reverse transcriptase, 5× RT buffer (4 μl) at 37 °C for 50 min, followed by 70 °C for 15 min, using a TaqMan Reverse-Transcription kit from Invitrogen.
A typical reaction was performed in 20 μl, consisting of 1 μl of cDNA and 10 μl of 2× SYBR Green I PCR mix, containing the specific primer pairs (final 10 pmol each). Denaturation was performed for 10 s at 95.0 °C, primer annealing for 10 s at 60 °C, and extension was performed for 10 s at 72.0 °C. Correct product size was confirmed by DNA agarose gel, and lack of primer dimer formation was verified by melt curve analysis, and the real time PCR products were sent to Shanghai Biosune Biotechnology Company (Shanghai, China) for sequence analysis. For real time PCR, comparing to the cornea, the samples with cDNA from the retina were used as the positive control, and the samples without cDNA were used as the negative control. Samples were analyzed in triplicate.
Western Blot: Western blot was performed as previously described [15]. For rho1 GABAC, we used the antibody as previously described [15]. For alpha1 GABAA and GABAB, we used the goat anti-human alpha1 GABAA and GABAB receptor polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The homology for the antibody sequence between chicken and human was more than 90% for the alpha1 GABAA (Homologene Blast comparison of human alpha1 GABAA, accession number: NP_001121120.1, with Gallus gallus alpha1 GABAA receptor, accession Number, P19150.1), and 100% for the GABAB (Homologene Blast comparison of human GABAB, accession number: NP_005449.5, with Gallus gallus GABAB receptor, accession Number, XP_419066.3).
Total protein was extracted separately from each tissue sample by lysing the cornea and retina in ice-cold lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), including 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS. Samples were centrifuged at 15,000× g at 4 °C for 15 min. The protein concentration was detected using BAC kits (Beyotime Institute of Biotechnology). Aliquots of protein extracts were loaded in each lane of 7.5% sodium dodecyl sulfate-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes for electrophoresis, and blocked in Tris Buffered Saline with Tween (TBST; 5% fat-free dry milk, 0.1% Tween-20, 150 mM NaCl, and 50 mM Tris at pH 7.5) for 2 h. The membranes were exposed to goat anti-human alpha1 GABAA, GABAB, and rho1 GABAC polyclonal antibody (Santa Cruz Biotechnology) at a 1:100 dilution in blocking buffer and incubated overnight at 4 °C. This was followed by incubation with a rabbit anti-goat secondary horseradish peroxidase (HRP)-labeled antibody (Zhongshan Goldenbridge Biotechnology Co. LtD. Beijing, China) at a dilution of 1:5,000 for 1 h at 37 °C. Protein bands were exposed to a negative film, developed, and fixed. The film was scanned and analyzed with Fluorchem TM 9900 Analyzer Software. β-actin (Zhongshan Goldenbridge Biotechnology Co. LtD.) was used as a housekeeping protein to normalize the protein load. Samples were analyzed in triplicate.
Immunohistochemistry: Immunohistochemistry was used to investigate the expression and distribution of alpha1 GABAA, GABAB, and rho1 GABAC receptors in chick cornea at the protein level, and was performed as has been described previously [15,38]. Whole eyes were freeze-mounted onto sectioning blocks. Vertical sections (8 μm thick) were cut from the anterior pole of the eye on a Leica RM2125 microtome (Leica Microsystems, Muenster, Germany) and thaw-mounted onto gelatin-coated glass slides. Sections were fixed for 10 min in acetone. Fixed sections were washed three times with PBS, covered with 10% BSA (Sigma, St Louis, MO) diluted in PBS, and incubated for 20 min at 37 °C. The slides were incubated at 4 °C overnight with primary antibodies (goat anti-human alpha1 GABAA, GABAB, and rho1 GABAC receptors polyclonal antibody, Santa Cruz Biotechnology) at a 1:50 dilution in blocking buffer. Sections were incubated in PBS without primary antibodies as a negative control. The antibody-treated and negative control sample slides were washed with PBS and exposed to a rabbit anti-goat secondary horseradish peroxidase (HRP)-labeled antibody (Zhongshan Goldenbridge Biotechnology Co. LtD.) at a dilution of 1:500 for 1 h at 37 °C. The slides were washed in PBS three times. A nickel solution of DAB (3,3′diaminobenzidine tetrahydrochloride; Zhongshan Goldenbridge Biotechnology Co. LtD.) was prepared and applied to the sections for less than 30 s. The DAB was then thoroughly rinsed from the sections using water. Sections were then stained with Hematoxylin and Eosin. The sections were dehydrated three times for 1 min in 100% ethanol, and then cleared in Histoclear (Huntz Biotechnology Co. LtD. Shanghai, China) three times for 1 min. The sections were examined with a light microscope (40×) and the images were digitized using a camera. Samples were analyzed in triplicate.

Alpha1 GABAA, GABAB, and rho1 GABAC receptor mRNA expression in chick cornea:
We observed the mRNA expression of alpha1 GABAA and rho1 GABAC receptors in all chick cornea and retina, but not in the negative control. We observed the mRNA expression of GABAB receptors only in chick retina, but not in the cornea and not in the negative control. Ethidium bromide-stained agarose gels of real time PCR products were positive for mRNA expression of alpha1 GABAA and rho1 GABAC receptors in chick cornea and retina, but not in the negative control run, and for mRNA expression of GABAB receptors only in samples of chick retina, but not in the cornea and negative control run. Products corresponding to alpha1 GABAA, GABAB, and rho1 GABAC receptors were amplified and expression occurred at the nucleic acid size marker base pair consistent with that of the alpha1 GABAA (255 bp), GABAB (217 bp), and rho1 GABAC (105 bp) receptors (Figure 1). The sequence analysis of the real time PCR products revealed that the sequence of the products corresponded to the targeted sequence of the mRNA of the alpha1 GABAA, GABAB, and rho1 GABAC receptors with the primers.
Alpha1 GABAA, GABAB, and rho1 GABAC receptor protein expression in chick cornea: Using antibodies for alpha1 GABAA receptors, one intense band (approximately 51 kDa) was detected in all chick corneal and retinal samples. Using antibodies for GABAB receptors, one intense band (approximately 130 kDa) was detected in the chick retina samples but was not present in the corneal samples. Using antibodies for rho1 GABAC receptors, one intense band (approximately 48 kDa) was detected in the chick corneal and retinal samples. Using antibodies for β-actin, one intense band (approximately 43 kDa) was detected in all the chick corneal and retinal samples ( Figure 2). Alpha1 GABAA, GABAB, and rho1 GABAC receptor localization in chick cornea: Alpha1 GABAA, and rho1 GABAC immunoreactivity was observed in chick corneal epithelium, was not observed in the stroma and not in the endothelium. No corneal layer displayed immunoreactivity to GABAB antibodies. In the retina, immunoreactivity for each of the antibodies was observed in the inner plexiform layer, outer plexiform layer, inner nuclear layer and ganglion cell layer, corresponding with previous published data [6][7][8][9][10][11]15]. There was essentially no immunoreactivity observed in the negative controls tissues (Figure 3).

DISCUSSION
Our results add new information on the distribution of GABA receptors within ocular tissues; GABAA receptors have been previously reported to be localized to a few cultured human corneal stem cells [5], here we clearly show that the chick cornea is positive for mRNA and protein specific for alpha1 GABAA and rho1 GABAC receptors, but negative for GABAB receptors. It would be of great interest to know if the corneas of other animal species also express GABA receptors and if so which sub-receptor types and in which corneal tissues. Staining density suggests that the alpha1 GABAA and rho1 GABAC receptors occur throughout the entire chick corneal epithelial layer, and are located on epithelial cells. The finding suggests a possible, but untested, role of the alpha1 GABAA and rho1 GABAC receptors in control of corneal functions.
Receptor types that are generally thought of as having primarily retinal localizations and function have been observed in corneal tissue. This includes muscarinic receptors [2,19,20,37], dopamine receptors [4], melatonin receptors [3], and retinoic acid receptors [18], here we show that the alpha1 GABAA and rho1 GABAC receptors, but not GABAB receptors are located in chick corneal epithelium. The functions mediated through these different receptors are diverse, eg sensation and epithelial cell proliferation (muscarinic receptors [19,20]), ion transport (dopamine receptors [39]), diurnal variations in corneal hydration and thickness (melatonin [40]), and maintenance of the ocular surface (retinoic acid receptors [18]).
Muscarinic receptor subtypes have been observed on bovine corneal epithelial cells [2], and on human corneal epithelium and endothelium [19,20]. The muscarinic receptor agonist, carbachol (0.001-100 microM) can increase the intracellular Ca 2+ concentration in bovine corneal epithelial cells, and if the cells were preincubated with either 1 microM atropine or 1 microM pirenzipine [2] this was suppressed. When rats were fed a muscarinic agonist, corneal opacities with histopathological features including neovascularization, acanthosis, and stromal proliferation were observed in a doserelated fashion at 100 and 200 mg/kg/day [41]. Dopamine receptors, the D2 subtype, are located in rabbit cornea epithelial and endothelial layers [4]. Dopamine increase Clsecretion by the activation of specific dopamine receptors, and stimulate ion transport within the rabbit corneal epithelium [39]. Melatonin receptors (Mel1a [40]) are expressed in chick corneal epithelium, stroma and endothelium. It has been suggested [40] that melatonin may modulate daily rhythms in corneal hydration/thickness via melatonin receptors on the corneal endothelium. Unlike dopamine, muscarinic and melatonin receptors that are localized in both epithelium and endothelium, here alpha1 GABAA and rho1 GABAC are only located in epithelium.
One of the important functions of the cornea is maintaining its optical transparency which is crucial for high quality visual performance. Corneal transparency is dependent on regulation of the hydration of the cornea, and the Clion channel is involved in fluid transportation within the corneal epithelium and endothelium [42][43][44]. The presence of Clion channels has been reported in human and rabbit corneal epithelium [45,46], in rabbit endothelium [45], and in rabbit corneal keratocytes [47,48]. GABA and its analogs have been shown to activate the rabbit corneal endothelial fluid pump, and this stimulation was abolished by the GABAA antagonists, bicuculline and chlorpromazine [16]. In this study we found alpha1 GABAA and rho1 GABAC receptors in chick corneal epithelium, but not the endothelium, and thus it seems unlikely that GABA is involved in regulating the fluid pumps, at least within the chick corneal endothelium. Whether alpha1 GABAA and rho1 GABAC receptors regulate Clconductance, and then influence transparency within the chick cornea requires further investigation. Determination of the effect of GABA agents on corneal hydration would resolve this.
To maintain corneal composition, organization and clarity the cornea has the greatest density of peripheral sensory nerves of any ocular tissue. Nerve terminals in the cornea are almost exclusively nociceptive Aδ and C fibers originating from the ophthalmic branch of the trigeminal ganglion [49], and structural and functional specialization of Aδ and C fiber free nerve endings innervate the corneal epithelium [50] across the entire corneal surface [1]. In this study we found alpha1 GABAA and rho1 GABAC receptor within the chick corneal epithelium. A potential role for these GABA receptors might involve regulating corneal sensitivity or other neural functions and this requires further investigation. With antibodies for alpha1 GABAA, GABAB and rho1 GABAC receptors, immunoreactivity was observed in corneal epithelium cell for alpha1 GABAA (α1) and rho1 GABAC (ρ1; yellow arrow), but not for GABAB receptor (B) in the cornea (C). In the retina (R), immunoreactivity was found in the inner plexiform layer, outer plexiform layer, inner nuclear layer, and ganglion cell layer for alpha1 GABAA (α1), GABAB (B) and rho1 GABAC (ρ1) receptors, corresponding to previous reports [6][7][8][9][10][11]15]. There was essentially no immunoreactivity observed for the negative controls (N.C). The scale bar is 10 µm. Photographs were taken at 40× magnification.
If the GABA receptors are to have a functional role within the cornea then there must be a supply of GABA either within the cornea, aqueous humor or tears to interact with the receptors. GABA-synthesizing enzyme was observed in the rat cornea [17], which implys that there is endogenous synthesis of GABA in rat cornea. It may be possible that cholinergic neurons within the cornea release GABA, like they do within retinal tissue [51]. We could not locate any articles that mention the presence of GABA within the eye's aqueous humor or tears. All of these potential sources of GABA are yet to be tested in the chick eye.
In conclusion, we found that both alpha1 GABAA and rho1 GABAC receptor were located within the chick corneal epithelium; the GABAB receptor was not present. The presence of GABA receptors within chick corneal epithelium suggests that some epithelial cell functions can be modified by GABA. Research is underway to determine how activation of these receptors alters corneal functions and where GABA or other substances that modifies them are expressed.